February 10, 2012

Dr. Ruth Lunn Director, Office of the RoC, DNTP National Institute of Environmental Health Sciences P.O. Box 12233 Research Triangle, NC 27709

Dear Dr. Lunn:

On behalf of the Flavor and Extract Manufacturers Association (FEMA), please find below additional information relevant to the nomination of alkenylbenzenes (selected dietary: estragole, myristicin, isosafrole) to the Report on Carcinogens.

Estragole was determined to be GRAS (Generally Recognized as Safe) by the FEMA Expert Panel in 1965 and is listed in the U.S. Code of Federal Regulations permitting use as a flavoring ingredient in food in the United States (21 CFR 172.515). In 2001, the FEMA Expert Panel re-evaluated the safety of methyl eugenol, a structurally related allyl alkoxybenzene, as a flavoring ingredient and concluded that:

…present exposure to methyl eugenol . . . resulting from consumption of food, mainly spices and added as such, does not pose a significant cancer risk. Nevertheless, further studies are needed to define both the nature and implications of the dose-response curve in rats at low levels of exposure to methyl eugenol . . .” (Smith et al., 2001).

The Joint FAO/WHO Expert Committee on Food Additives (JECFA) recently evaluated a group of allyl alkoxybenzenes, including methyl eugenol, present in foods and essential oils and used as flavouring agents (JECFA, 2009a). Their conclusions are summarized below:

The Committee concluded that the data reviewed on the six alkoxy-substituted allylbenzenes provide evidence of toxicity and carcinogenicity to rodents given high doses for several of these substances. A mechanistic understanding of these effects and their implications for human risk have yet to be fully explored and will have a significant impact on the assessment of health risks from alkoxy-substituted allylbenzenes at the

concentrations at which they occur in food. Further research is needed to assess the potential risk to human health from low-level dietary exposure to alkoxy-substituted allylbenzenes present in foods and essential oils and used as flavouring agents.

Below we have summarized ongoing research to address the JECFA’s and the FEMA Expert Panel’s recommendations for further research. This research includes biochemical studies investigating the metabolic activation and detoxication of the reactive metabolites of methyl eugenol and its close structural relative, estragole, and the possible matrix effects in methyl eugenol and related alkoxy-substituted allylbenzene-containing naturals (e.g. basil oil) (see Biochemical Studies below). Research has also been undertaken to investigate the relationship of dose to the initiating effects for carcinogenesis (see Short-term Studies below). Although both of these research programs are not yet completed, the preliminary results are relevant to the Report on Carcinogens consideration of estragole, myristicin and isosafrole. Both programs are expected to be completed by the end of 2012.

Also presented is a short summary of a recent publication applying the margin of exposure (MoE) approach to methyl eugenol.

IOFI and FEMA would be pleased to continue to discuss this ongoing research program with the National Toxicology Program. Please contact us for further information and explanation as needed.

Sincerely,

Timothy B. Adams FEMA Senior Scientist

Sean V. Taylor FEMA Scientific Director

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Introduction

The metabolic fate of a compound is determined by numerous factors including chemical

structure, species, gender dose and genetic factors to name the most important. Above all,

chemical structure, i.e. the presence of particular functional groups that are subject to

metabolism, will largely determine the overall metabolic fate of a compound and quite minor

structural variations may elicit major consequences for metabolic outcomes and events linked to

this such as toxicity. In the present paper this is further evaluated in a detailed manner for a

group of structurally related hydroxy- and alkoxy- substituted alkyl- and propenylbenzenes.

Hydroxy- and alkoxy- substituted allyl- and propenylbenzenes are important constituents of a

variety of botanical based food items. These classes of compounds vary from one another by the

presence or absence of alkylation of their p-hydroxyl substituents and/or the position of the

double bond in the alkyl side chain. The present paper provides an overview of how these subtle

structural variations dominate the metabolism of these important food-borne compounds

ultimately influencing their toxicity and especially their genotoxic and carcinogenic potential. It

is concluded that whereas the p-alkoxyallylbenzenes safrole, methyleugenol and estragole

undergo metabolic activation to produce genotoxic carcinogens, replacement of their p-alkoxy

substituents by a hydroxyl moiety and/or isomerisation of the alkyl chain double bond alters the

metabolic fate of these important naturally occurring flavour molecules in favour of

detoxification pathways, thereby eliminating bioactivation to a genotoxic carcinogenic

metabolite.

Biochemical Studies

The metabolic fate of a compound is determined by numerous factors, the most noteworthy

including chemical structure, species, gender, dose and genetic factors. Above all, chemical

structure, i.e. the presence of particular functional groups that are subject to metabolism, will

largely determine the overall metabolic fate of a compound. Although the metabolic options for a

variety of structural functional groups are well understood, nevertheless, at times, quite minor

structural variations may elicit major consequences for metabolic outcomes and events linked to

this such as toxicity. The capacity of minor structural changes to influence metabolic outcomes

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can be illustrated by two examples. The metabolism and toxicity of the organocyanide (nitrile)

function is determined by its structural attachment. When attached to an aliphatic function it is

metabolised to generate hydrogen cyanide and such aliphatic nitriles that are highly toxic

(Williams, 1959; Potter et al., 2001; Willhite and Smith, 1981). However, if the nitrile function is

bonded directly to an aromatic ring it is metabolically stable and the organonitrile is

characterised by low toxicity (Tanii and Hashimoto, 1984). Small changes in chemical structure

can dramatically influence the patterns of metabolic conjugation of carboxylic acids.

Furthermore, the simple carboxylic acid, benzoic acid, is metabolised in humans by conjugation

with glycine to form hippuric acid (Bridges et al., 1970; Caldwell, et al., 1980). However, the

next homologue, phenylacetic acid, is metabolised along a quite different pathway, involving

conjugation with glutamine to form phenacetylglutamine (James et al., 1972). In addition, if the

phenylacetic acid acquires an alpha-methyl function (hydratropic acid) then the capacity to form

amino acid conjugates is completely lost and this acid is metabolised by conjugation with

glucuronic acid (Dixon et al., 1977)). In this way, quite subtile structural variations can have a

major influence on metabolic and toxicity outcomes. The present paper further provides a

detailed evaluation for a group of structurally related hydroxy- and alkoxy-substituted alkyl- and

propenylbenzenes.

Hydroxy- and alkoxy- substituted allyl- and propenylbenzenes are important constituents

of a variety of botanical-based food items. Examples of food-borne compounds belonging to

these classes are the allylalkoxybenzenes estragole, methyleugenol and safrole, the propenyl

alkoxybenzenes anethole (4-methoxypropenylbenzene) and isoeugenol methylether (3,4-

dimethoxypropenylbenzene), the allyl hydroxybenzenes chavicol (4-hydroxyallylbenzene) and

eugenol (4-hydroxy-3-methoxyallylbenzene) and the propenyl hydroxybenzenes isochavicol and

isoeugenol (4-hydroxy-3-methoxypropenylbenzene). The chemical structures of these related

compounds are presented in Figure 1. Table 1 presents an overview of the occurrence of these

compounds in the modern food chain.

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Allyl Propenyl Allyl Propenyl alkoxybenzenes alkoxybenzenes hydroxybenzenes hydroxybenzenes

O OH

estragole anethole chavicol isochavicol

O OH

O OO O O OH O OH

isoeugenolmethyl methyl eugenol eugenol isoeugenol ether

O

O

safrole

Figure 1. Chemical structures of allyl- and propenylalkoxy- and hydroxybenzenes.

The allylalkoxybenzenes safrole, methyleugenol and estragole are important constituents

of herbs like nutmeg, cinnamon, anise star, tarragon, sweet basil, sweet fennel and anise vert, and

are present in their essential oils as well as in food product derived from these botanicals such as

pesto. These compounds have been documented to be hepatotoxic and genotoxic leading to liver

tumors at high dose levels in rodent bioassays (Drinkwater et al., 1976; Miller et al., 1983; Wiseman

et al., 1987; SCF 2001a,b,c; Smith et al., 2002). The related compounds eugenol, chavicol,

isoeugenol, isoeugenol methyl ether and anethole, are food-borne structural analogues in which

the number of alkylated hydroxyl moieties varies and/or the double bond in the alkyl side chain

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O O

O

Methyl eugenol Estragole 3,4-dimethoxyallylbenzene 4-methoxyallylbenzene

is shifted. Although the principal target organ for the toxicity of all these compounds is the liver,

the structural analogues do not share the genotoxicity and resulting carcinogenicity of safrole,

methyleugenol and estragole. The present paper presents an overview of how these subtle

structural changes impact the ultimate hazard potential of these important flavour molecules.

Recent studies evaluated the effect of dietary factors on the metabolic activation and

detoxication of reactive metabolites (i.e., 1’-sulfoxy metabolite) formed from methyl eugenol. In

vitro studies have been performed to elucidate the various reactions involved in the 1’-

hydroxylation, sulfation, and glucuronidation pathways of allylalkoxybenzene derivatives such

as methyl eugenol. The effect of animal species and chemical structure on the involved enzyme-

catalyzed reactions has been the major focus of most recent research. A very close structural

relative to methyl eugenol is estragole (Figure 2).

Figure 2. The chemical structures of methyl eugenol and estragole, two alloxyallylbenzenes that

are metabolized via similar mechanisms.

Estragole is a natural constituent of several herbs and spices including sweet basil. In

rodent bioassays, estragole induces hepatomas, an effect ascribed to estragole bioactivation to 1′-

sulfooxyestragole. In vivo it has been suggested that the sulfate ester of 1’-hydroxyestragole

undergoes cleavage to form a reactive carbocation that, at sufficient concentration, forms protein

and DNA adducts. The flavone, nevadensin (Figure 3), a basil constituent, has been identified as

an inhibitor of DNA adduct formation in rat hepatocytes exposed to the proximate carcinogen 1′-

hydroxyestragole (Alhusainy et al., 2010). Nevadensin has been reported to be a major flavone

in basil (Ocimum basilicum) comprising 5.6-58.4% of total flavones present (Grayer et al.,

1996).

6

O O

HO O

O

OH O

Figure 3. The chemical structure of Nevadensin (5,7-dihydroxy-6,8,4’-trimethoxyflavone).

The IC50 of nevadensin in primary rat hepatocytes is reported to be 0.16 ± 0.09 µM.

Neither nevadensin nor 1’-hydroxyestragole demonstrated cytotoxicity as would be indicated by

increased lactate dehydrogenase activity in culture supernatant. Primary rat hepatocytes exposed

to 50 µM 1’-hydroxyestragole in the presence of increasing concentrations of nevadensin (0-4

µM) showed a concentration dependent decrease in DNA adduct formation (Alhusainy et al.,

2010). The lack of cytotoxicity in addition to the DNA adduct inhibition activity suggests that

nevadensin is able to pass through the cell membrane to exert its effects. This inhibition occurs at

the level of sulfotransferase (SULT)-mediated bioactivation of 1′-hydroxyestragole. The non-

competitive inhibition constant (Ki) for SULT inhibition by nevadensin was 0.004 µM in male

rat and human liver fractions. Concentrations of nevadensin up to 20 µM did not inhibit 1′-

hydroxyestragole detoxication by glucuronidation and oxidation. This was demonstrated by no

significant effect upon Km and Vmax values in the presence or absence of nevadensin in 1’-

hydroxyestragole incubated with primary rat or human hepatocyte S9 fractions (Alhusainy et al.,

2010).

The inhibition of SULT by nevadensin was incorporated into the recently developed

physiologically-based biokinetic (PBBK) rat and human models for estragole bioactivation and

detoxification. The results predict that co-administration of estragole at a level inducing hepatic

tumors in vivo (50 mg/kg bw) with nevadensin at a molar ratio of 0.06, representing the ratio of

their occurrence in basil, results in almost 100% inhibition of the ultimate carcinogen 1′-

sulfooxyestragole when assuming 100% uptake of nevadensin. Assuming 1% uptake, inhibition

would still amount to more than 83%. Altogether these data point at a nevadensin-mediated

inhibition of the formation of the ultimate carcinogenic metabolite of estragole, without reducing

the capacity to detoxicate 1′-hydroxyestragole via glucuronidation or oxidation. These data also

point at a potential reduction of the cancer risk when estragole exposure occurs within a food

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matrix containing SULT inhibitors compared to exposure to pure estragole (Alhusainy et al.,

2010).

The carcinogenicity of allylalkoxybenzenes has been well-documented. According to

NTP researchers, for example, clear evidence of hepatocellular adenomas, hepatocellular

carcinomas and hepatoblastomas has been repported in B6C3F1 mice administered 37, 75, or

150 mg/kg bw /day of methyleugenol by gavage (NTP, 2000). The profile of responses was

similar in rats with an increased incidence of hepatocellular adenomas and carcinomas reported

in both male and female F344/N rats at dose levels (gavage) of 37, 75, 150, and 300 mg/kg bw/

day of methyl eugenol. An increased incidence of hepatocholangiomas and

hepatocholangiocarcinomas were also reported at the two highest dose levels in male rats (NTP,

2000). Similar results were obtained at the NTP in a 13-week oral toxicity study, when rats and

mice were dosed with 37.5, 75, 150, 300 or 600 mg/kg bw per day of estragole via corn oil

gavage (NTP, 2011). The genotoxic and hepatocarcinogenic action of safrole, methyleugenol and

estragole have been correlated to formation of their proximate carcinogenic 1’-hydroxy

metabolites followed by sulfation to the ultimate carcinogenic 1’-sulfoxy metabolites (Borchert

et al., 1973; Wislocki et al., 1976; Wislocki et al., 1977; Wiseman et al., 1985; Drinkwater et al.,

1976; Miller et al., 1976). Figures 4 and 5 provides a schematic overview of the major metabolic

pathways for the allylalkoxybenzenes including this bioactivation route as well as several

detoxification pathways. A major detoxification pathway of the 1’-hydroxy metabolites of the

allylalkoxybenzenes in rats are conjugation with glucuronic acid catalysed by UDP-

glucuronosyltransferases (UGTs), resulting in a stable metabolite which is excreted in the urine

(Stillwell et al., 1974; Benedetti et al., 1974; Solheim, 1973; Anthony et al., 1987). In humans,

however, oxidation of the 1’-hydroxy metabolite of estragole to the ketone appeared to be the

major pathway for detoxification of 1’-hydroxyestragole (Punt et al., 2009), which appears to be

a minor pathway in rats (Punt et al., 2008). Recent results reveal that these species-specific

differences in glucuronidation and oxidation also exist for 1’-hydroxymethyleugenol and 1’-

hydroxysafrole (Al-Subeihi et al., submitted; Martati et al. submitted).

In the absence of a free phenolic function, O-dealkylation is an important detoxification

pathway that competes favourably with 1’-hydroxylation of safrole, methyleugenol and

estragole, especially at low dose levels in all species, particularly humans and mice (Zangouras

et al., 1981; Sangster et al., 1983; Anthony et al., 1987). However, as dose levels increase, two

8

+

O -

O3SO

R

O

R= -H, -OH or -OCH3

Red arrows indicates intoxication

Green arrows indicate detoxication

Short arrows indicate low dose pathway

Long arrows indicate high dose pathways R

DNA and/or Protein Adducts

Allyl alkoxybenzenes

OO HO HO

rodent pathway human pathway

RRR R R O OOO O

O-Demethylase epoxidation

UDP-glucuronosyl transferase

SG

O GlcO

R

OH RR R O O UDP-glucuronosyl O

transferase

Epoxide hydrolase/ SGGlutathione-S-transferase

OH

RR

OGlc O

Figure 4. General routes of detoxication and intoxication for allyl alkoxybenzene flavor ingredients

9

R= -H, -OH or -OCH3

Red arrows indicates intoxication

Green arrows indicate detoxication

Short arrows indicate low dose pathway

Long arrows indicate high dose pathways

O-Demethylation

R

OH

UDP-glucuronosyl transferase

Propenyl alkoxybenzenes

DNA and/or Protein Adducts

O HO

SG epoxidation Glutathione-S-

R transferase

O R R O O

3'-Hydroxylation Epoxide hydrolase

OH OH

OH

ROH R O

O

OH

R

O

R OH OGlc

R

OOH

O NHCH2CO2H

R

O R

O Glutathione-S-

transferase

OH

GS

R

OH

Figure 5. General routes of detoxication and intoxication for propenyl alkoxybenzene flavor ingredients

10

bioactivation pathways compete favorably with O-demethylation. One pathway leads to an

epoxide of the allyl double bond. This epoxide has been shown to be able to form DNA adducts

in vitro, but these adducts are not found in vivo (Luo and Guenther, 1996). These results have

been ascribed to the rapid detoxification of the epoxide by epoxide hydrolases and/or

glutathione-S-transferases in vivo (Luo et al. 1992; Luo and Guenthner, 1995, 1996; Guenthner

and Luo, 2001; Ioannides et al., 1981; Klungsøyr and Scheline, 1983). The other pathway, 1’-

hydroxylation, discussed above, becomes a prominent pathway in rodents at dose levels >10

mg/kg bw/day (Zangouras et al., 1981; Anthony et al., 1987). As dose levels increase, saturation

of the O-demethylation pathway and utilisation of the 1’-hydroxylation pathway provide

conditions for increased formation of the downstream genotoxic metabolites that are proposed to

elicit hepatocarcinogenic effects in mice and rats (Benedetti et al., 1977). The sulphate conjugate

of the 1’-hydroxylation metabolite is currently believed to be the proximate carcinogenic metabolite

for this class of compounds (Wiseman et al., 1987; Phillips et al., 1981; Phillips et al., 1984;

Randerath et al., 1984; Wiseman et al., 1985). The sulphate conjugate may react with DNA, RNA

and proteins but can also be detoxified through conjugation with glutathione. As a result only a

fraction of the 1’-sulfoxy metabolite formed is expected to result in DNA adducts. Upon reaction of

1′-sulfooxyestragole with DNA, several adducts can be formed (Figure 6). These adducts include

N2-(trans-isoestragole-3′-yl)-deoxyguanosine (E-3’-N2-dG), N2-(estragole-1′-yl)-

deoxyguanosine (E-1′-N2-dG), 7-(trans-isoestragole-3′-yl)-deoxyguanosine (E-3’-7-dG), 8-

(trans-isoestragole-3′-yl)-deoxyguanosine (E-3’-8-dG), and N6-(trans-isoestragole-3′-yl)-

deoxyadenosine (E-3′-N6-dA) (Phillips et al., 1981; Punt et al., 2007). The major adduct formed

with the guanine base is N2-(trans-isoestragole-3′-yl)-deoxyguanosine (E-3′-N2-dG). Recently it

has been reported that adducts between estragole and adenine, N6-(trans-isoestragol-3′-yl)-

deoxyadenosine (E-3′-N6-dA) (E-3′-N6-dA) may also be formed to a significant extent in the

liver of male rats (F344) exposed to estragole at a dose level of 600 mg/kg bw for 4 weeks (Ishii

et al., 2011). The structure of these adducts is in accordance with formation of 1’-

sulfoxyestragole and the resonance stabilisation of its reactive intermediate via isomerisation of

the C2’-C3-double bond to the C1’-C2’ position.

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O N

N

NH

O

OH OH

NH

N

CH

CH3

O NN

H

OH OH

NH

N

O N

N

NH2

O

OH OH

NH

N

CH2

CHCH

CH CH

CH2

O N

N

NH2

O

OH

NH

N

adduct minor addduct minor adduct minor adduct OH

OMe

OMe OMe OMe OMe

HCCH

HReaction with C N

H2+ adenine base NNCH2C C

CH HO H CH OSO3- H CHHC

CH NN

CH2 OCH2 CH2 CH2

Estragole 1'-hydroxyestragole 1'-sulfooxyestragole Carbocation E-3'-N6-dAOHReaction with OH

guanine base

OMe

CHCH

CH2

OMe

N

O

OMe OMe

majorE-3'-N2-dG E-1'-N 2-dG E-3'-8-dG E-3'-7-dG

Figure 6. Structures of allylalkoxybenzene purine base adducts.

Thus, the biotransformation of these substances are dose-dependent, species- and sex-specific. At

low doses in rodents and humans, alkylated aromatic ring substituents are converted to phenolic

derivatives that can be excreted either unchanged or as conjugates (Zangouras et al., 1981; Sangster

et al., 1983, 1987; Anthony et al., 1987; Beyer et al., 2006). At similar doses, O-dealkylation

appears to be more favoured in the mouse and human than in the rat (Zangouras et al., 1981;

Sangster et al., 1983; Anthony et al., 1987). At higher dose levels, including those commonly used

in rodent toxicity and carcinogenicity studies, biotransformation of these compounds involves

greater oxidation of the allyl side chain (i.e., 1’-hydroxylation and alkene epoxidation) (Zangouras

et al., 1981; Anthony et al., 1987). As such, when groups of rats are given increasing oral dose

levels of safrole (0.9, 60 or 600 mg/kg bw), a dose-dependent change from O-demethylenation

metabolites to allyl side chain 3’-hydroxylation (Figure 5) and/or formation of eugenol or its isomer

occurs especially when the dose is raised from 60 to 600 mg/kg bw (Benedetti et al., 1977). In a

similar study with estragole, as the dose is increased (from 0.05 to 5, 500 or 1000 mg/kg bw) in

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mice and rats, the relative extent of O-demethylation decreases with a parallel increase in 1’-

hydroxylation, especially at 50 and 1000 mg/kg bw/day (Zangouras et al., 1981; Anthony et al.,

1987). The metabolic switching to a metabolic activation pathway is more significant in rats

compared to mice at higher dose level (Phillips et al. 1981; Swanson et al., 1981; Miller et al.,

1983; Wiseman et al., 1985). A previously developed physiologically based kinetic (PBK) model

for estragole bioactivation and detoxification in male rat (Punt et al., 2008) corroborated that a shift

from O-demethylation at lower doses to 1'-hydroxylation at higher doses occurs. O-

demethylation of estragole was shown to be the major metabolic route at low doses of estragole,

occurring mainly in the lung and kidney of male rat. In these organs, 4-allylphenol is formed

with high affinity, reflected by a relative low Km. Due to saturation of the O-demethylation

pathway in the lung and kidney, formation of the proximate carcinogenic metabolite 1'-

hydroxyestragole, which was shown to occur mainly in the liver of male rat, becomes relatively

more important at higher doses of estragole.

The effect of dose and metabolic switching on formation of the 1’-hydroxy metabolite in

rats and humans provides additional insight into the carcinogenic potential of allylalkoxybenzene

derivatives in both rats and humans. Rats show a clear dose-dependent increase in 1’-

hydroxysafrole or its downstream metabolites in the urine. Humans show no evidence of such

metabolites at low dose (1.66 mg; ~0.03 mg/kg bw) (Benedetti et al., 1977). At increasing doses

(0.05, 5.0, 50 mg/kg bw) rats excrete 0.9%, 3.6%, and 5.5% 1’-hydroxy metabolites,

respectively, whereas human volunteers given a 100 ug dose excrete only 0.3 % of the dose in

the form of the 1’-hydroxy metabolite (Zangouras et al., 1982). Recent results revealed that this

may be due to an important species difference in the way 1’-hydroxyestragole is converted in rat

and human (Punt et al., 2009). In human 1’-hydroxyestragole is mainly converted to 1’-

hydroxyglucuronide which can be detected in urine. In human however the major route for

conversion of 1’-hydroxyestragole appeared to be oxidation to 1’-oxoestragole, indicating

urinary analysis should focus on other urinary metabolites. In spite of this species difference in

the pathway for detoxification of 1’-hydroxyestragole, the level of formation of 1’-

sulfoxyestragole appeared to vary less than 2-fold between rat and human (Punt et al., 2009).

This overview of the metabolic pathways of methyleugenol and its related p-

alkoxyalkylbenzenes reveals that bioactivation and generation of the genotoxic metabolite

requires a 2,3-unsaturated allyl side chain providing an activated carbon (C1) that is both allylic

13

and benzylic. The allyl side chain allows for formation of the proximate carcinogenic 1’-hydroxy

metabolite and the subsequent formation of the ultimate carcinogenic 1’-sulfoxy metabolite. Also

a para alkoxy substituent (OCH3) on the phenyl ring is required to stabilize the incipient reactive

intermediate that forms from the 1’-sulfoxy metabolite. The overview also suggests that once O-

dealkylation converts an alkoxy substituent into a hydroxyl moiety, possibilities for swift

conjugation and excretion may occur. The next sections evaluate to what extent modifications in

the allyl side chain and/or the alkoxy subtituents in the allylalkoxybenzenes modify their

metabolic fate and ultimate toxicological effects.

Modification of the allyl side chain: metabolism and toxicity of anethole and isoeugenol

methyl ether

Isomerisation of the double bond in the allyl side chain of methyleugenol and estragole

gives rise to isoeugenol methyl ether and anethole, respectively (Figure 1). Extensive

biochemical and toxicological data (Newberne et al., 1999) document that the target organ for

anethole toxicity is the liver and that the ultimate hepatotoxic metabolite is anethole epoxide.

Anethole epoxide exhibits hepatocellular cytotoxicity at concentrations 10 times lower than

those of anethole itself (Bounds and Caldwell, 1996; Caldwell, 1992; Marshall and Caldwell,

1992, 1996; Howes et al., 1990). Inhibitors of detoxification enzymes for anethole epoxide (i.e.,

epoxide hydrolase and glutathione transferase) increase the cytotoxic effects of anethole

(Marshall and Caldwell, 1992). From toxicity studies, species and sexes that show the highest

rate of daily production of anethole epoxide also exhibited signs of hepatotoxicity at the lowest

dose levels of anethole (Newberne et al., 1989, 1999). Since neither anethole nor anethole

epoxide are genotoxic (Nestmann et al., 1980; Heck et al., 1989; Mortelmans et al., 1986;

Gorelick, 1995; Howes et al., 1990; Marshall and Caldwell, 1992, 1996; Calwell et al., 1992;

Randerath et al., 1984; Phillips et al., 1984; Abraham et al., 1996; Marzin et al., 1979; Al-Harbie

et al., 1995), the hepatotoxic effects and neoplastic effects observed at high dose levels in studies

with laboratory animals result from a non-genotoxic mechanism in which toxicity is the result of

continuous elevated hepatocellular concentrations of anethole epoxide. Quantitative data on the

conversion of anethole to anethole epoxide can be related to hepatotoxic effects observed in

these short-term and long-term rodent studies (Truhaut et al., 1989).

14

Figure 4 presents an overview of the metabolic pathways for anethole. Metabolic studies

have shown that at low dose levels (<5 mg/kg bw/day) trans-anethole is rapidly absorbed and

detoxified in rodents primarily by O-demethylation and in humans primarily by 3’-

hydroxylation- (Bounds and Caldwell, 1992, 1996; Sangster et al., 1987; Caldwell and Sutton,

1988). In humans, efficient detoxification of trans-anethole by 3’-hydroxylation and subsequent

β-oxidation yields conjugated benzoic acid derivatives which are rapidly excreted in the urine

(Sangster et al., 1987; Caldwell and Sutton, 1988). At these dose levels, bioactivation and

toxicity via epoxidation is a minor pathway and is quantitatively similar among mice, rats, and

humans with the rat showing a slightly greater tendency to epoxidise compared to the mouse or

human.

From this overview it follows that the isomerisation of the double bond in the allyl side

chain shifts the regioselectivity of the cytochrome P450 mediated hydroxylation of the chain

resulting in a 3’-hydroxy metabolite instead of a 1’-hydroxymetabolite. This not only eliminates

the chances of formation of the proximate carcinogenic metabolite, but also shifts the

metabolism to a detoxification pathway, because the 3’-hydroxymetabolite readily undergoes

hydroxyl group oxidation followed by β -oxidation that yields conjugated benzoic acid

derivatives which are rapidly excreted in the urine (Sangster et al., 1987; Caldwell and Sutton,

1988).

The inclusion of an additional methoxy substituent at the meta-position in isoeugenol

methyl ether increases metabolic options for detoxification which reduces endogenous levels of

the corresponding epoxide (Solheim and Scheline, 1976). Male albino Wistar rats administered a

single oral dose of 200 or 400 mg isoeugenyl methyl ether/kg bw formed greater than 77%

urinary metabolites principally via 3- and 4-O-demethylation and 3’-hydroxylation. Metabolites

resulting from the epoxidation pathway account for less than 1% of the urinary metabolites. The

low production of the corresponding epoxide correlates with the lack of reported hepatotoxic

effects for isoeugenyl methyl ether.

Taken together, the data suggest that at low levels of exposure, trans-anethole and

isoeugenyl methyl ether are efficiently detoxicated in rodents and humans. Chronic exposure to

high dose levels in female rats, results in the continuous daily production of hepatocellular

concentrations of anethole epoxide that induce a continuum of cytotoxicity, cell proliferation,

cell death, and following prolonged exposure, liver tumors. Quantitatively, daily conversion of

15

anethole to the epoxide in 2 year study of female rats (Truhaut et al., 1989) resulted in an

increase in the incidence and severity of hepatotoxicity was in the range of 30 to 120 mg/kg bw

per day (15% epoxide metabolites at an anethole dose of 200 mg/kg bw per day and 23%

epoxide metabolites at anethole dose of 550 mg/kg bw/day) (Newberne et al., 1999; Bounds,

1994). At the highest dietary level of anethole (550 mg/kg bw per day) female rats exhibited both

hepatic toxicity and hepatic tumors, but at dietary levels of 120 mg/kg bw per day of anethole

(predicted to generate approximately 22 mg anethole epoxide/kg bw per day), there was no

evidence of hepatotoxicity. In female rats showing no hepatotoxic effects, the lifetime body

burden to anethole epoxide was approximately 17,000 mg/kg while in humans, lifetime body

burden of anethole epoxide resulting from intake of anethole (0.002 mg/kg bw per day) is

approximately 51 mg/kg. These numbers reveal a large margin between exposure levels causing

hepatotoxicity in experimental animals and levels expected to occur in realistic human exposure

scenarios.

Thus, comparison of the metabolic and toxicity profile of anethole and isoeugenyl methyl

ether to that of the related allylalkoxybenzenes safrole, methyleugenol and estragole, reveals that

the isomerisation of the double bond in the allyl side chain significantly alters the chemical

characteristics and thus the metabolism favors detoxification, eliminating formation of the

proximate genotoxic 1’-hydroxymetabolite and resulting in a different mode of toxic action.

Modification of the alkylation state of the hydroxyl substituents: metabolism and toxicity of

chavicol and eugenol

O-Dealkylation of a methoxy substituent in methyleugenol and estragole gives rise to eugenol

and chavicol, respectively (Badger et al., 1999; Zangouras et al., 1981; Figure 1). Thus, the

difference in toxicity between methyleugenol and eugenol can only be associated with the

presence of a free hydroxyl moiety in eugenol. The free phenolic function implies that

conjugation becomes a primary mode of metabolism and excretion. Figure 2 presents the

metabolic pathways for allylhydroxybenzenes, also representative for eugenol, and reveals that

additionally eugenol undergoes 1) isomerization to yield isoeugenol that may then undergo 3’-

hydroxylation and reduction of the double bond, 2) epoxidation of the allyl double bond to yield

an epoxide that hydrolyzes to the corresponding diol which then may be oxidized to the

corresponding lactic acid derivative, 3) conjugation of its quinone-methide type intermediate

16

with glutathione, and 4) hydroxylation at the allyl position to yield 1’-hydroxyeugenol. Since all

of these metabolites possess a free phenolic OH group or other polar oxygenated functional

groups, they readily conjugate with glucuronic acid or sulphate and are excreted in the urine

(Fischer et al., 1990). In human experiments, 95% of an ingested dose of 0.6 mg or 150 mg

eugenol was excreted in conjugated form in the urine within 24 hours (Sutton, 1986; Fischer et

al., 1990). Within 24 hours, greater than 55% of a 150 mg oral dose of eugenol administered in

gelatine tablets after consumption of a normal breakfast was excreted in the urine as the

glucuronic acid or sulphate conjugates of eugenol. Other conjugated urinary metabolites included

cis- and trans-isoeugenol (7%) formed by isomerization of the double bond, 3-(4-hydroxy-3-

methoxyphenyl)propane formed by reduction of either eugenol or isoeugenol, and 3-(4-hydroxy-

3-methoxyphenyl)propionic acid (4.6%) presumably formed by allylic hydroxylation of

isoeugenol followed by NADH-dependent enzymatic reduction of the double bond. Conjugated

metabolites formed from epoxidation of eugenol included eugenol epoxide (1.6%), the

corresponding diol (3%), and 2-hydroxypropionic acid derivative, 3-(4-hydroxy-3-

methoxyphenyl)propionic acid (3.3%) formed by oxidation of the diol primary alcohol.

Tentatively identified metabolites included a thiophenol (11%) metabolite presumably formed by

glutathione conjugation at an aromatic ring position and a trace amount (<1%) of 1’-

hydroxyeugenol from by allylic hydroxylation of the benzylic position. The authors concluded

that eugenol is subject to rapid first pass conjugation and rapid elimination accompanied with

only a small fraction participating in isomerization, epoxide-diol, glutathione conjugation or 1’-

hydroxylation pathways (Fischer et al., 1990).

At lower dose levels, conjugation predominates. Over 85% of the urinary radioactivity of

0.6 mg dose of [14C]-eugenol given to two volunteers was excreted after 24 hours in the urine as

glucuronic acid and sulfate conjugates of eugenol with the glucuronic acids predominating.

Essentially all (95 and 103%) of the radioactivity was excreted in 24 hours. Minor amounts (2%

each) of the corresponding diol 3-(4-hydroxy-3-methoxyphenyl)propane-1,2-diol and alcohol 3-

(4-hydroxy-3-methoxyphenyl)propane-2-ol were also detected (Sutton, 1986). Results of studies

in rodents indicate that eugenol has a metabolic fate similar to that in humans. In a dose-

dependent metabolism study, female Wistar rats were administered 0.5, 5, 50 or 1000 mg/kg bw

of ring labelled [14C]-eugenol in trioctanoin by stomach tube. At 24 hours, the urinalysis revealed

glucuronic acid and sulphate conjugates of eugenol, the O-demethylation metabolite 3,4-

17

dihydroxypropylbenzene and the reduced metabolite 3-methoxy-4-hydroxypropylbenzene

(Sutton et al., 1985; Sutton, 1986). At low doses (0.5, 5, and 50 mg/kg bw), sulphate conjugates

were the principal metabolites while at the highest dose (1000 mg/kg bw) glucuronic acid

conjugates predominated. Metabolites resulting from reduction or O-demethylation (i.e., 3,4-

dihydroxypropylbenzene and 3-methoxy-4-hydroxypropylbenzene) were not detected at the high

dose in the 24-hour urine.

As outlined above, based on experiments in humans, it can be concluded that the majority

of orally administered eugenol is conjugated with glucuronic acid or sulphate and excreted in the

urine. Minor metabolic pathways include oxidation to form the corresponding epoxide followed

by hydrolysis to the diol and then further oxidation products; isomerization followed by allylic

oxidation and then reduction; reduction of the alkene; or 1’-hydroxylation (trace). All these

metabolites possess a free phenolic hydroxy moiety and are readily conjugated and excreted in

the urine.

The increased possibilities for swift conjugation and excretion due to the free phenolic

hydroxyl moiety can explain why eugenol is not a genotoxic carcinogen, in contrast to

methyleugenol (NTP, 2000; Maronpot et al., 1987). In studies with eugenol, there was no

significant increase in the incidence of hepatocellular neoplasms in either male or female

B6C3F1 mice in 2-year bioassay study at any dose level (Maronpot et al., 1987) and there was no

evidence of an increased incidence of hepatocellular neoplasms in male or female F344/N rats

(NTP, 1983) at dietary levels calculated to provide a daily intake of 150, 300, or 625 mg

eugenol/kg bw/ day (FDA, 1993). The swift detoxification of eugenol, due to its free phenolic

hydroxyl, is entirely consistent with a lack of any significant toxicologic and carcinogenic

responses. The short residence time of eugenol in the body explains the lack of toxic effects

observed in numerous studies on eugenol.

Modification of both the allyl side chain and the alkylation state of the hydroxyl

substituents: metabolism and toxicity of isoeugenol

Combined isomerisation of the allyl side chain and replacement of the p-methoxy substituent by

a para-hydroxyl moiety converts methyleugenol to isoeugenol (Figure 1). In isoeugenol the

presence of a hydroxyl substituent at the para-position in combination with the shift in the

double bond in the allyl side chain modifies the metabolic options and resulting toxicity

18

compared to methyleugenol. Conjugation of the free phenolic function with sulfate and

glucuronic acid is the predominant detoxification pathway for isoeugenol. Greater than 85% of a

156 mg [14C]-isoeugenol/kg bw administered by gavage to male Fischer-344 rats was excreted as

the glucuronic acid and sulfate conjugate in the urine within 72 hours (Badger et al., 1999).

Likewise, 82% was excreted as the same urinary conjugates 72 hours after 15.6 mg [14C]-

isoeugenol/kg bw, administered by intravenous injection. Following both administration routes,

approximately 10% of the administered dose was excreted in the faeces and less than 0.1% was

recovered as expired CO2 with less than 0.25% of the dose remaining in selected tissues.

Although the 2-year NTP bioassay (NTP, 2010) in mice showed some evidence of

increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular

adenoma or carcinoma (combined) at oral gavage doses of 75, 150, or 300 mg/kg bw/ day of

isoeugenol. This response was not dose-related and was only reported in male B6C3F1 mice, a

sex and species with historically high levels of background hepatocellular neoplasms (Maronpot

et al., 1987). From a biological perspective, the increase in the tumor incidence in male B6C3F1

mice reflects the impact of high-dose liver damage to an organ already prone to spontaneous

development of liver neoplasms (Haseman et al., 1986, 1989). The lack of any hepatotoxic or

hepatocarcinogenic effects in female mice or either rat sex support the conclusion that

isoeugenol does not present a carcinogenic hazard to humans when consumed at regular dietary

intake levels estimated to be 45 µg/day in Europe and 26 µg/day in the USA which is equivalent

to 0.75 and 0.43.mg/kg bw/day in Europe and the USA respectively (EFFA, 2004; Gavin et al.,

2008). And the lack of toxicity even at relatively high levels of exposure is due to conjugation,

3’-hydroxylation, and β -oxidation pathways that provide efficient routes of detoxification

reducing the toxic potential as compared to that of methyleugenol.

Although the 2-year NTP bioassay (NTP, 2010) in mice, showed some evidence of increased

incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or

carcinoma (combined) at oral gavage doses of 75, 150, or 300 mg/kg bw/ day of isoeugenol, the

response was not dose-related and it was reported only in male B6C3F1 mice, a sex and species

with historically high levels of background hepatocellular neoplasms (Maronpot et al., 1987).

From a biological perspective, the increase in the incidence of tumors in male B6C3F1 mice

reflects the impact of high-dose liver damage to an organ already prone to spontaneous

development of liver neoplasms (Haseman et al., 1986, 1989). The lack of any hepatotoxic or

19

hepatocarcinogenic effects in female mice or male or female rats support the conclusion that

isoeugenol does not present a carcinogenic hazard to humans when consumed at regular dietary

intake levels and the lack of toxicity even at relatively high levels of exposure is due to

conjugation, 3’-hydroxylation, and β -oxidation pathways that provide efficient routes of

detoxification reducing the toxic potential as compared to that of methyleugenol.

Short-term Studies

Although it has been demonstrated (JECFA, 2009b) that a linear relationship exists

between DNA adduct formation and dose level of methyl eugenol, no comprehensive study of

the relationship of dose to the initiating effects for carcinogenesis have been investigated. To

study initiating effects of methyleugenol in rat liver, groups of male F344 rats (8/group) were

administered methyl eugenol by intragastric instillation in 0.5% methylcellulose, at dosages of 0,

(C, control groups 1 and 2), 62 (LD, low-dose groups 3 and 4), 125 (MD, mid-dose groups 5 and

6), or 250 (HD, high-dose groups 7 and 8) mg/kg bw/dose, 3 doses per week for 8 and 16 weeks

(equivalent to dosages of 37, 75 or 150 mg/kg bw/day, 5 days per week as in the National

Toxicology Program; see table 1). At 8- and 16-week intervals of initiation, 3 rats from each

dose level (groups 1, 3, 5 and 7) were killed and samples of liver and blood (16 weeks only)

were taken for analysis of blood liver enzymes, DNA adducts, cell proliferation (liver only) and

hepatocellular altered foci (HAF, liver only) (Private Communication to IOFI, 2010).

Table 1. Comparative cumulative doses

14-Week study a 16-Week study b

Individual doses Cumulative doses Individual doses Cumulative doses

(mg/kg bw/dose) (mg/kg) (mg/kg bw/dose) (mg/kg)

10 700 NA NA

30 2100 62 3000

100 7000 125 6000

300 21000 250 12000

1000 70000 NA NA a, NTP, 2000; Abdo et al., 2001; Herbert and Abdo, 2003: The daily doses (5 doses per week) were 10, 30, 100, 300, or

1000 mg/kg bw in this 14-week study with male and female rats. This amounted to 50, 150, 500, 1500 or 5000 mg/kg

bw/wk. 7000 mg/kg produced 1/10 hepatocellular adenoma in one male rat; from this study 185, 375 or 750 mg/kg bw/wk

were chosen as the doses of the bioassay;

20

b, present study: Based on the above study, the 3 doses were selected, i.e., 62, 125 or 250 mg/kg bw/dose, 3 doses per wk, or

185, 275 or 750 mg/kg bw/wk;

NA, not applicable.

No compound-related deaths or clinical findings occurred in the study. No changes were

evident in any of the serum liver enzymes monitored at the 16-week interval.

At all 3 dose levels administered, methyl eugenol produced a reduction in body weight

gain, which was statistically significant (p=0.005) at 8 weeks in the two highest doses. This

significant reduction represented 12% and 21% of the MD and HD, respectively. The reduction

in body weight gain and relative liver weight increase (p=0.011) is in accordance with the

literature for similar cumulative doses (NTP, 2000; Abdo et al., 2001; Herbert and Abdo, 2003).

Moreover, the consequences of these 2 findings led to hypoproteinemia (Abdo et al., 2001),

which was not monitored in the present study. In addition, with protein catabolism of this

magnitude, i.e., 21% decrease in body weight gain at 8 weeks, a shift in energy homeostasis is

usually produced in which, after exhaustion of carbohydrates, fat cannot be utilized as an energy

substrate, especially in the liver (Matthews and Battezzati, 1994; Williams and Iatropoulos,

2002). Nevertheless, by week 16, equilibrium was reestablished, as reflected by the fact that the

mean body weight gains and terminal mean body weights had recovered.

At 8 weeks, in MD and HD groups there were group mean body weight gain reductions

(p=0.005), group mean body weight reductions (p=0.005), and increases in group mean relative

liver weight (p=0.011). 32P-Nucleotide postlabelling (NPL) revealed 3 adducts (2 major and one

minor) in the livers of all dosed groups. The mean hepatic DNA-adduct value of the MD and

HD groups were significantly increased compared to control and LD groups. Proliferating cell

nuclear antigen (PCNA) immunohistochemistry revealed that the mean group hepatocellular

replicating fraction (RF) in all methyl eugenol dosed groups was increased (p<0.01) compared to

controls, as was the hepatocellular hypertrophy, karyomegaly and multinucleation, Kupffer cell

activation, and enlargement of the periportal cholangiole plexus. Only in the HD rats were HAF

identified by placental-type (π) glutathione s-transferase (GST-P) immunohistochemistry.

At 16 weeks, there were no body weight changes, or terminal body weight changes when

compared to controls, respectively. All methyl eugenol groups had dose-related increases

(p=0.05 to p=0.005) in mean DNA-adduct values in both liver and WBCs compared to controls,

although the WBC levels were approximately 100 times less than the liver. The mean group 21

hepatocellular RF in all methyl eugenol groups was significantly (p<0.001) increased compared

to controls, as was the incidence and severity of the diffuse hepatocellular hypertrophy,

karyomegaly and multinucleation, Kupffer cell activation, and enlargement of the periportal

cholangiole plexus. All methyl eugenol groups had HAFs in a dose-related pattern.

Three DNA adducts, two major and one minor, were found in the liver, even in the LD

group. However, not having reference standards, it was not possible to say with certainty that

these DNA adducts were identical to those reported in cultured human hepatocytes (Zhou et al.,

2007), or F344 rat liver (Ellis, 2007). At 8 weeks, the mean liver DNA adduct values of MD and

HD groups were statistically higher (p<0.001) compared to both groups 1 (control) and 2 (LD

methyl eugenol). This is the first time that DNA adducts are reported with adult F344 rat livers.

At 16 weeks, the LD and MD methyl eugenol groups were significantly elevated (p=0.05), with

the HD group assuming a significance at p=0.005. In addition, the DNA adduct levels were

higher. With WBC at 16 weeks, likewise all 3 methyl eugenol groups were significantly

elevated compared to controls. Here the adduct levels present were about 1% of the adducts

found in the liver. This may be a useful biomarker, as WBC DNA can be readily obtained and

monitored in humans.

The low levels of DNA adducts in control samples probably reflects exposure of controls,

which is known to occur (Samsone and Fox, 1977; Nicholls et al., 2005). This could be a

consequence of the relative volatility of methyl eugenol, despite the fact that the control cages

had a special cover.

The mean RF values of all 3 methyl eugenol groups were significantly increased in a

dose-and-time-related pattern, compared to controls. Moreover, comparing the 2 interim

sacrifice mean RF values of all methyl eugenol groups, the 16-week values were lower,

indicating that the hepatocytic compensatory (adaptive) response was completed and was

receding. This is again similar to the BrdU-monitored hepatocellular proliferation response

(Abdo et al., 2001; Herbert and Abdo, 2003). The recession of proliferation suggests that with

further dosing methyl eugenol would have negligible promoting activity through this mode of

action.

At both sacrifice intervals, the methyl eugenol liver findings, using hematoxylin and

eosin and GST-P histopathology comprised several dose-and-time-related changes. A diffuse

hepatocellular hypertrophy, with sporadic karyomegaly and binucleation, with concomitant

22

activation of Kupffer cells with pigment was present. Also, in periportal fields, there was an

enlargement of the cholangiole plexus expanding the canals of Herring, which give rise to

cholangiocytes-lined cholangioles. In the PCNA stained slides the PCNA positive

cholangiocytes and neighboring pluripotential oval cells were evident in a dose-related

increasing pattern, while they were absent in the controls. This reflects an adaptive

compensatory response to increased hepatobiliary metabolic demands resulting from methyl

eugenol dosing because the rat does not have a gall bladder to gauge the intrahepatic biliary

homeostasis (Williams and Iatropoulos, 2002; Greaves, 2007). Thus, the enlarged plexus is not

the same as the "bile duct hyperplasia", which was described previously, at comparable

cumulative doses (Abdo et al., 2001; Herbert and Abdo, 2003). Bile duct hyperplasia can be

viewed as a preneoplastic precursor of hepatobiliary neoplasia (e.g. furan, NTP, 1993), but this

neoplasia did not occur in the 2-year methyl eugenol study (NTP, 2000).

Starting with the 8-week interval, the HD methyl eugenol rats had mixed foci of altered

hepatocytes (HAF), which were GST-P positive. The incidence of GST-P positive HAF value

was 0.51 ± 0.3, and the severity was mild. This incidence value was significant (p<0.01)

compared to all other groups. At 16 weeks, the incidence and severity of HAF was increased in

a dose-related manner in all methyl eugenol groups. The incidence of GST-P positive values of

1.26 ± 0.24 was significantly (p<0.01) increased compared to all other groups. The average

diameter of each HAF lesion has increased from ~ 204 µ in the LD and MD groups, to ~ 918 µ in

the HD group. This is in accordance with the previously published literature (Abdo et al., 2001;

Herbert and Abdo, 2003). The induction of HAF reflects development of promotable

preneoplastic lesions, which we expect will be promoted with phenobarbital in the promotion

phase (for 24 weeks) of this study. However, compared to an activation-dependent DNA-

reactive hepatocellular carcinogen, 2-acetylaminofluorene (AAF), at both carcinogenic and

noncarcinogenic doses (with PB promotion) in male F344 rats, the GST-P positive HAF values

at 12 weeks in the HD group at a cumulative dose of 12000 mg/kg methyl eugenol were 15 times

lower with methyl eugenol compared to AAF given at a cumulative dose of 94 mg/kg (Williams

et al., 1998; 2004). At this phase of the study, the initiating activity of methyl eugenol can be

considered weak.

Thus, biomarker changes of initiation were produced by 8 and 16 weeks of dosing with

methyl eugenol. One half of the remaining groups (3, 5, 7) of rats are currently being exposed to

23

500 ppm phenobarbital, in the diet, to promote hepatocellular neoplasia at 24 weeks of dosing,

with the other half of the groups (6, 4, 8) being on recovery for 24 weeks.

At 8 weeks, the high dose methyl eugenol rats only had mixed foci of altered hepatocytes

(HAF), which were GST-P positive. The GST-P incidence was 0.51 ± 0.3 and the severity was

minimal, with an average size of ~204µ. At 16 weeks, the incidence and severity of HAF was

increased in a dose-related manner in all methyl eugenol groups. The incidence of GST-P

positive HAF value of 1.26 ± 0.24 in the high dose group, was significantly (p< 0.01) increased

compared to low and mid dose groups. Also, the severity in the high dose group increased from

minimal to mild, and the average size from ~204 to ~ 918µ. This phenomenon is in accordance

with the previously published literature (Abdo et al., 2001; Herbert and Abdo, 2003). Based on

these data and other data from this laboratory, the following conclusions can be made:

There is a clear dose response for the development of GST-P foci and positive

hepatocellular altered foci (HAF)/cm2 from the liver tissue of rats treated for 8 and 16

weeks. The induction of HAF reflects development of promotable preneoplastic lesions,

which, it is anticipated, will be promoted to tumors with Phenobarbital (PB) in the

promotion phase (now on week 35).

Compared to an activation-dependent DNA-reactive hepatocellular carcinogen, 2-

acetylaminofluorene (AAF), at both carcinogenic and non-carcinogenic doses (with PB

promotion) in male F-344 rats, the GST-P positive HAF values at 16 weeks in the methyl

eugenol HD group at a cumulative dose of 12000 mg/kg methyl eugenol were 15 times

lower with methyl eugenol compared to AAF given at a cumulative dose of 94 mg/kg at

12 weeks (Williams et al., 1998; 2004).

Thus, the initiating activity of methyl eugenol can be considered weak.

Application of the Margin of Exposure (MoE) approach to Methyl Eugenol

Smith et al., recently applied the margin of exposure (MoE) approach to substances in

food that are genotoxic and carcinogenic using methyl eugenol as an example. The MoE

approach relies on the comparison of benchmark dose levels (BMDL) for carcinogenicity with

different estimates of human intake. Based on all models applied, the BMD for a 10% incidence

24

of male rat liver adenomas and carcinomas (combined) is 7.9 mg/kg-bw/day. Using the estimated

human daily intakes of 10 µg/kg-bw/day (Smith et al., 2002) up to a maximum theoretical intake

of 66 µg/kg-bw/day for high consumers (EFSA, 2004), MoE of 800 and 100 were determined.

The authors noted the MoE values were extremely low and further elaborated on the caveats

influencing MoE results, specifically metabolic shifting, possible matrix effects from intake of

methyl eugenol and related alkoxy-substituted allylbenzenes and the “nature and implications of

the dose response curve in rats a low levels of exposure” (Smith et al., 2010). The authors also

recommended additional studies were needed and noted the complexity in the reflection of these

caveats in a numerical MoE (Smith et al., 2010).

Conclusions

In this review, a mechanism-based approach is used to present an example illustrating

how minor structural changes may change metabolic pathways for bioactivation and

detoxification in such a way that overall toxicity is significantly affected. This was demonstrated

by comparing the metabolic fate and toxicologic sequelae of a series of naturally occurring

food-borne substances that vary in structure only in the position of the double bond on the

alkenyl side chain or the presence of a para-hydroxy or para-methoxy functional group. The

impact of subtle structural changes on the mode of action for the four types of substances

demonstrates that a mode-of action-based approach, in which the relationship between chemical

structure and ADME characteristics leading to either preferential detoxification or formation of a

bioactivated metabolite, can be used to interpret the potential pathologic, genotoxic and

carcinogenic effects of these related food-borne natural compounds. It is concluded that

isomerization of the double bond in the allyl side chain or the presence of a free phenolic

hydroxyl moiety shifts the metabolism in favor of detoxification and eliminates the possibilities

for formation of a genotoxic carcinogenic metabolite. This explains the remarkable differences in

the toxicity of these related hydroxy- and alkoxy- substituted allyl- and propenyl-benzenes, and

illustrates how subtle structural changes can have significant effects on toxicity.

In a second study of methyl eugenol, the initiating of hepatic tumor formation was evaluated in

an 8 and 16-week study using phenobarbital as a tumor promoter. Although in the LD group, there

was evidence of DNA adducts, cell proliferation (RF), and hepatocellular altered foci (HAF), there

25

was no evidence of initiation to hepatocellular adenomas. This lack of tumorigenic response occurred

at a daily dose of 62 mg/kg bw/dose or a cumulative dose of 3000 mg/kg. At this level, there was no

increase in hepatocellular adenomas with or without PB promotion. Thus, the dose level of 62 mg/kg

bw/dose, represents a NOAEL dose for neoplasia, which is not capable of producing promotable

hepatocellular neoplastic induction, under these experimental conditions, in spite of the presence of

DNA adducts, increased RF and HAF. Identification of a lower dose that does not induce these

effects would contribute to the risk assessment of methyl eugenol. In conclusion, methyl eugenol

exhibits a low carcinogenic potential compared to that of 2-acetylaminofluorene and present no

significant risk to humans under current conditions of use.

26

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